Omega Engineering 30, 48, 20, 25 manual Some frequently asked questions

11. Some frequently asked questions

Q1 What is the ‘Pause’ function at the start of the program used for?

A1 Some users prefer to preheat the heated lid before placing the samples into the unit. The pause feature is used to stop the unit a fter the 4 minut e heated lid preheat step. It will also sound an audible alarm indicating that the machine is ready for the sample tubes or pla te to be added. Pressing the “Pause” key followed by the “Enter” key will commence the remaining program.

Q2 Why do you have to press some keys twice to make the unit do what you want it to do?

A2 With many of the program f eatures there is a fail-safe function which asks if you are sure you want to perform a particular action. You are required to press one key to select a function followed by a second to accept it.

Q3 What is ‘Hot Start’?

A3 A “hot start” is used for reducing non-specific product for mation during the first cycle of amplification. The ‘Hot S tart’ programming step is used to pause the instrument at a specific temperature, typically around 70ºC, after the initial template denaturation. The reason is to allow the manual addition of unmodified Taq DNA polymerase which may loose activity if added during the initial 5min denaturation. Heat-activated Taq or Hot Start enzy mes do not require this step.

Q4 What is the incremental function? (See page 24).

A4 Incremental timing and temperature are used to increase or decrease either the time or temperature incrementally over the number of cycles in a stage. Incrementation of extension time is used with ‘Long PCR’ which is when large template fragments are to be amplified (e.g. 27kb lambda DNA, 40kb ge nomic DNA).

Decremental temperature is used for protocols such as ‘Touchdown PCR’ where one starts with a high annealing temperature in the first cycle and gradually decreases the temperature over the numbe r of cycles in the stage. This ensures that only the specific product is amplified.

Q5 The heat-up ramp-rate in the specification is 3.6°C/second yet I can only manually set it to 2.0°C/second. Why is this?

A5 If you set the ramp-rate to max the block will heat at 3.6°C/second the unit sets its own control parameters. If you program a ramp-rate the control parameters reduce the ramp-rate. This is a feature of the software and not the actual ramp- rate.

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